Folate metabolism and physiology continue to represent attractive targets for chemotherapeutic intervention. This is clearly demonstrated by the recent development of new classes of folate analogues targeted to folate- dependent biosynthetic reactions that have entered clinical trial. The applicant's own work with his collaborators has resulted in the design of a new 4-amino-folate analogue, edatrexate, with significantly improved therapeutic activity against certain solid tumors in patients compared to methotrexate in advanced clinical development. The studies proposed extend this work and seek further improvement in the treatment of human cancer with new, more efficacious folate analogues, new or optimized regimens of therapy utilizing tumor-specific targeting or seeking synergistic combinations and by circumvention of acquired resistance. The underlying basis for these approaches stems from the applicant's identification in tumors of a specific route of mediated membrane transport and a distinct folylpolyglutamate synthetase that mediate 4-aminofolyl polyglutamate accumulation that are major determinants of therapeutic selectivity of these analogues. New studies will pursue in depth knowledge of these determinants in parental and antifolate-resistant tumor cells at the level of their functional and structural biochemistry, energetics and regulation of gene expression. The latter addresses the unique ontogenetics of these determinants in tumor cells compared to their functional counterparts in normal proliferative tissues that are sites of drug limiting toxicity for these antifolates. Work will also continue in tumor cells on the ATP- dependent efflux route for folate analogues and on folyl and antifolyl polyglutamate transport and turnover in lysosomes, focusing on the molecular properties of the transport system in each case. The mechanism of activation of lysosomal folyl polyglutamate hydrolase by endogenous reduced sulfhydryl compounds will also be examined. All of these studies present further opportunities for modulation therapies that achieve favorable intratumoral pharmacokinetics for new folate analogues including those targeted at sites other than DHFR.